Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga.

Monoterpenoid indole alkaloids are a large (∼3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here, we used next-generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC-MS analysis, confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.

Quantification of Anti-Addictive Alkaloids Ibogaine and Voacangine in In Vivo- and In Vitro-Grown Plants of Two Mexican Tabernaemontana Species.

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti-addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse-grown plants, in vitro-grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.